Anti-tumor effects of low-dose metronomic vinorelbine in combination with alpelisib in breast cancer cells

In metastatic breast cancer (MBC), PIK3CA mutations, activating the phosphatidylinositol 3-kinase (PI3K) signaling pathway seem to be associated with chemotherapy resistance and poor outcome. Inhibition of the PI3K signaling pathway may lead to sensitization and prevention of the development of resistance to cytotoxic drugs. The present study aimed to investigate the anti-tumor activity of low-dose vinorelbine (VRL) combined with alpelisib, an α-selective PI3K inhibitor and degrader, in breast cancer (BC) cells. Human BC cell lines MCF-7, T-47D [both hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated], MDA-MB-231 and BT-549 (both triple-negative, wild-type PIK3CA) were exposed to a combination of low-dose VRL and alpelisib for 3 and 7 days. Cell viability was detected by the Alamar blue assay, and cell proliferation was determined by the BrdU incorporation. The effect of the substances on the p110α protein expression that is encoded by PIK3CA gene was investigated by Western blot. Low-dose VRL plus alpelisib showed synergistic anti-tumor effects and significantly inhibited cell viability and proliferation of MCF-7 and T-47D cells. Even lower alpelisib concentrations (10 ng/ml and 100 ng/ml) combined with low-dose metronomic VRL led to a significant reduction of cell viability of PIK3CA-mutated cells, and the anti-tumor activity was comparable with the effects at 1000 ng/ml alpelisib. Cell viability and proliferation of MDA-MB-231 and BT-549 cells were inhibited by VRL but not by alpelisib alone. This indicates that alpelisib did not significantly affect the cell growth of triple-negative, PIK3CA wild-type BC cells. The p110α expression was downregulated or not affected in PIK3CA-mutated cell lines, and not significantly upregulated in PIK3CA wild-type cell lines. In conclusion, combination of low-dose metronomic VRL and alpelisib showed synergistic anti-tumor effects and significantly inhibited the growth of HR-positive, HER2-negative, PIK3CA-mutated BC cells, providing a rationale for further efforts to evaluate this combination in vivo.


Raw data of Figure 1a
Cell viability of MCF-7 cells exposed to low-dose vinorelbine and alpelisib for 3 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1b
Cell viability of MCF-7 cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1c
Cell viability of T-47D cells exposed to low-dose vinorelbine and alpelisib for 3 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1d
Cell viability of T-47D cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1e
Cell viability of MDA-MB-231 cells exposed to low-dose vinorelbine and alpelisib for 3 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1f
Cell viability of MDA-MB-231 cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1g
Cell viability of BT-549 cells exposed to low-dose vinorelbine and alpelisib for 3 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 1h
Cell viability of BT-549 cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 2
Cell viability of MCF-7, T-47D, MDA-MB-231 and BT-549 cells exposed to DMSO for 7 days. The data given are absolute values obtained from the Alamar blue assay.

Raw data of Figure 3a
Cell proliferation of MCF-7 cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the BrdU incorporation.

Raw data of Figure 3b
Cell proliferation of T-47D cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the BrdU incorporation.

Raw data of Figure 3c
Cell proliferation of MDA-MB-231 cells exposed to low-dose vinorelbine and alpelisib for 7 days. The data given are absolute values obtained from the BrdU incorporation.

Raw data of Figure 4a-4d
Cell viability of MCF-7, T-47D, MDA-MB-231 and BT-549 cells exposed to low-dose vinorelbine and alpelisib alone for 3 and 7 days. The data are shown as percentages relative to those of the untreated control cell culture. The absolute values of the Alamar blue assay are displayed in the tables of raw data of Figure 1.

Raw data of Figure 4e and 4f
Cell proliferation of MCF-7, T-47D and MDA-MB-231 cells exposed to low-dose vinorelbine and alpelisib alone for 7 days. The data are shown as percentages relative to those of the untreated control cell culture. The absolute values of the BrdU incorporation are displayed in the tables of raw data of Figure  3.

Raw data of Figure 6a
The p110α expression in the MCF-7 and T-47D cells after 7 days of treatment with low-dose vinorelbine plus alpelisib was quantified by densitometry evaluation using a computer-based pixel counting system (AlphaView, ProteinSimple, San Jose, CA, USA). These values were referenced to the β-actin values of the same membrane as a loading control.

Raw data of Figure 6b
The p110α expression in the MDA-MB-231 and BT-549 cells after 7 days of treatment with low-dose vinorelbine plus alpelisib was quantified by densitometry evaluation using a computer-based pixel counting system (AlphaView, ProteinSimple, San Jose, CA, USA). These values were referenced to the β-actin values of the same membrane as a loading control.